Interaction of dinitrophenyl-pepstatins with human cathepsin D and with anti-dinitrophenyl antibody. Development of potential reagents for the localization in vivo of active proteinases at sites of tissue injury.
Extracellular cathepsin D has been observed by various cytochemical methods at sites of tissue injury. However, the role of this enzyme in connective tissue matrix degradation is uncertain because there are no histochemical methods for determining whether or not the cathepsin D is active at such sites in living tissues. We considered that the combined use of a labelled tight-binding inhibitor with immunoprecipitation of the enzymes might overcome this problem. We have explored the application of derivatives of the inhibitor pepstatin, as only active cathepsin D binds pepstatin tightly. A series of N-pepstatinyl-N'-dinitrophenyl-alpha, omega-diaminoalkanes were synthesized with alkyl-chain lengths of two, four and six carbon atoms. These compounds were tight-binding inhibitors of human cathepsin D. In fluorescence-quenching titrations the dinitrophenyl groups were also fully available to bind high-affinity anti-dinitrophenyl antibody. It was shown by immunodiffusion in gels and by gel permeation chromatography that N-pepstatinyl-N'-dinitrophenyl-1,6-diaminohexane was a bifunction inhibitor able to bind cathepsin D and anti-dinitrophenyl antibody at the same time.
D000915:Antibody Affinity; D000937:Antigen-Antibody Reactions; D002402:Cathepsin D; D002403:Cathepsins / Q000037:antagonists & inhibitors*; D002850:Chromatography, Gel; D004136:Dinitrobenzenes / Q000276:immunology / Q000494:pharmacology; D005779:Immunodiffusion; D046911:Macromolecular Substances; D009842:Oligopeptides / Q000494:pharmacology*; D010436:Pepstatins / Q000276:immunology / Q000494:pharmacology*; D011485:Protein Binding
D004136:Dinitrobenzenes; D046911:Macromolecular Substances; D009842:Oligopeptides; D010436:Pepstatins; D002403:Cathepsins; D002402:Cathepsin D